Southern Blight of Perennial Swallowwort (Vincetoxicum spp.) in New York

Pale swallowwort [Vincetoxicum rossicum (Kleopow) Barbar.] and black swallowwort [Vincetoxicum nigrum (L.) Moench] are invasive perennial viny milkweeds that have become prevalent across natural and managed habitats in northeastern North America. Southern blight of V. rossicum caused by the fungus, Athelia rolfsii (Curzi) C. C. Tu & Kimbr., was reported at a New York county park in 2008, resulting in a decline in V. rossicum stands. The disease outbreak and persistence of the pathogen highlighted the potential of A. rolfsii for Vincetoxicum spp. control. To better characterize A. rolfsii’s pathogenicity and biology, we studied virulence to adult Vincetoxicum spp., spatiotemporal attributes of the Southern blight epidemic at the discovery site over four years, and sclerotial survival over two years. Disease incidence and severity were high for both Vincetoxicum spp. in misting chamber experiments. The spatiotemporal spread patterns of Southern blight in V. rossicum suggest the epidemic in the first year of monitoring (2016) was already highly aggregated and that subsequent spread was limited and resulted in significant local aggregation. Sclerotial survival studies at two locations (Pittsford and Ithaca, New York) demonstrated the A. rolfsii isolates can overwinter in upstate New York and are pathogenic to Vincetoxicum spp. the subsequent season. However, shallow burial of sclerotia more rapidly reduced survival compared with placement on the soil surface. Overwinter survival of A. rolfsii sclerotia in New York is notable as this pathogen is typically associated with sub-tropical and tropical regions. Broadcast applications of the pathogen would be needed for widespread Vincetoxicum control at a site, but even restricting releases to select locations would not prevent pathogen movement off-site via water or machinery. The known risks of the A. rolfsii isolate to other broadleaf plants in natural and agricultural settings suggest a low feasibility of use for the biological control of Vincetoxicum spp.

First report of Southern blight, caused by Athelia rolfsii (syn. Sclerotium rolfsii Sacc.), on Hellebores in North America

Lenten rose (Hellebores hybridus) is an herbaceous perennial grown in landscapes and valued for early spring flowers and high levels of deer resistance. An additional benefit as a landscape plant comes from the high level of disease resistance, with only three fungal pathogens reported in North America. In August of 2021, a Lenten rose plant within a mature landscape in Silver Spring, MD, USA, (lat 39.116629 long 77.043198) was found with a collapsed canopy and brown stems near the soil line. Small clusters of brown sclerotia-like objects were seen along the stem. Samples of the sclerotia and diseased tissue were dipped in 70 percent ethanol for 15 sec, transferred to 5 percent NaClO for 30 sec, immersed in sterile water for one minute, then plated onto Potato Dextrose Agar. Sclerotia-like objects germinated and white mycelia covered the plates within five days of germination. Hyphae emerged from diseased tissue within two days and also grew rapidly. Cultures from sclerotia-like objects and diseased tissue produced white sclerotia which melanized to brown spherical sclerotia ranging in size from two to four mm. Culture samples (1 cm square) were excised from the culture plates and transferred to the base of three two-year old potted hellebore plants. Control plants had blocks of PDA placed at the base of the plants. Plants were placed in plastic bags for two days to maintain humidity, then maintained at room temperature without plastic bags. Petioles turned brown and collapsed within seven days of inoculation. White, fan-like hyphae were present along with maturing sclerotia. Samples from surface sterilized tissue and sclerotia produced the same culture morphology as the originally isolated cultures. Non-inoculated plants remained healthy, and the pathogen was not isolated from non-inoculated plants. Individual DNA samples were prepared from original cultures and the re-isolated cultures. Molecular identification was performed by amplification of the internal rRNA transcribed spacer region (ITS1/4, White et al. 1990 ), the large subunit rRNA (LSU), and the elongation factor-1A (EF1a). Amplification products were cloned into TOPO-TA pcr4 vector and sequenced (Macrogen USA). Sequences were submitted to GenBank for IT1/4 (OK172559) and LSU (OK172560). Homology to ITS1/4 was found with Athelia rolfsii (MN622806), to LSU with Athelia rolfsii (MT225781) and for EF1a with Athelia rolfsii (MW322687). This is the first report of Athelia rolfsii on Hellebores in North America (Farr, D.F & Rossman, A.Y. Fungal Databases, U.S. National Fungus Collections, ARS, USDA. Retrieved September 10, 2021). This report is unique in that few pathogens are known to infect Hellebores(Taylor et al. 2011) and southern blight is not commonly isolated in landscape plantings at Maryland latitudes. 1. White et al. PCR Protocols: A Guide to Methods and Amplifications. Academic Press, San Diego, 1990 2. Taylor, R.K., Romberg, M.K. & Alexander, B.J.R. A bacterial disease of hellebore caused by Pseudomonas viridiflava in New Zealand. Australasian Plant Dis. Notes 6, 28–29, 2011.

First Report of Southern Blight Caused by Sclerotium delphinii on Euonymus fortunei in China

Euonymus fortunei is an evergreen shrub-vine in the family Celastraceae, widely used as a groundcover or a vine to climb walls, or traditional herbal medicine in China. In August 2019, typical southern blight symptoms that included basal stem rot and the presence of sclerotia in rotted tissue were observed on E. fortunei in Kunshan city, Jiangsu province, China. Disease incidence was estimated at approximately 15 to 20%; meanwhile, approximately 30 to 40% of diseased plants died. The infected plants showed brown to dark stem necrosis near the base, leaf yellowing and wilting. White mycelia and white to dark reddish-brown sclerotia were observed at the base of the stem and rotten tissue. To isolate the causal organism, infected stem tissue and sclerotia collected from diseased plants in a median strip in Kunshan (31°23'40"N, 120°54'57"E) were disinfected with 70% ethanol for 2 to 3 sec, followed by 2 min in 5% NaClO, rinsed three times with sterile water, then plated on potato dextrose agar (PDA) medium, and incubated at 25°C. Isolated colonies were subcultured by needle tip transfer 3 days later. Isolates had white mycelia on PDA, with a radial growth rate of 15.2 to 18.7 mm/day. White and orange sclerotia were developed after 5 to 8 days and eventually turned dark reddish-brown. The sclerotia were globoid or irregular with surface markings (1.4 to 4.3 mm diam.; mean = 2.59 mm; n = 50) on PDA, and the average number of sclerotia produced per Petri dish ranged from 35 to 85 (mean = 52; n = 10). Microscopic observations found septal hyphae and clamp connections. These morphological features were identical to the description of Sclerotium delphinii (syn. Sclerotium rolfsii var. delphinii) (Mukherjee et al. 2015; Punja and Damiani 1996; Stevens 1931). A representative isolate YKY2020.01 was stored in the Key laboratory of National Forestry and Grassland Administration on Ecological Landscaping of challenging Urban Sites in Shanghai. For molecular identification, DNA of the isolate YKY2020.01 was extracted using the Fungal DNA Kit (OMEGA bio-tek, China). The internal transcribed spacer region (ITS fragment including ITS1, 5.8S rDNA, and ITS2 region) was amplified with primers ITS1/ITS4 (White et al. 1990), and then sequenced by Sangon Biotech (Shanghai, China). BLAST analysis in NCBI found the ITS sequence of YKY2020.01 (MW916955) was 99.84% similar to S. delphinii strain CBS272.30 (MH855140). Phylogenetic analysis using maximum likelihood (ML) method placed isolate YKY2020.01 in the same clade as S. delphinii. To evaluate pathogenicity, hyphal blocks (0.7 cm diam.) were placed at the base of the stem of healthy E. fortunei (n = 5 plants). Five healthy plants were inoculated by uncolonized agar blocks as controls. All plants were kept in a greenhouse with a temperature range from 21 to 25.6°C (mean = 24.9°C) and relative humidity of 50%. Inoculated plants were symptomatic after 3 days and wilted after 12 days. Symptoms in inoculated plants were similar to those observed under natural conditions, whereas the control group remained asymptomatic. The fungal pathogen was reisolated from symptomatic tissue and confirmed as S. delphinii. To the best of our knowledge, this is the first report of S. delphinii causing southern blight on E. fortunei in China and worldwide. This finding provides concise and practical information on the newly emerged disease of E. fortunei, which is beneficial for future disease management. References: Mukherjee, A. K., et al. 2015. J. Plant Pathol. 97:303. Punja, Z. K. and Damiani, A. 1996. Mycologia 88:694. Stevens, F. L. 1931. Mycologia 23:204. White, T. J., et al. 1990. Page 315 in PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA. Funding: This work was supported by the Key Project of Science and Technology Commission of Shanghai Municipality (19DZ1204102).

JA signal-mediated immunity of Dendrobium catenatum to necrotrophic Southern Blight pathogen

Background Dendrobium catenatum belongs to the Orchidaceae, and is a precious Chinese herbal medicine. In the past 20 years, D. catenatum industry has developed from an endangered medicinal plant to multi-billion dollar grade industry. The necrotrophic pathogen Sclerotium delphinii has a devastating effection on over 500 plant species, especially resulting in widespread infection and severe yield loss in the process of large-scale cultivation of D. catenatum. It has been widely reported that Jasmonate (JA) is involved in plant immunity to pathogens, but the mechanisms of JA-induced plant resistance to S. delphinii are unclear. Results In the present study, the role of JA in enhancing D. catenatum resistance to S. delphinii was investigated. We identified 2 COI1, 13 JAZ, and 12 MYC proteins in D. catenatum genome. Subsequently, systematic analyses containing phylogenetic relationship, gene structure, protein domain, and motif architecture of core JA pathway proteins were conducted in D. catenatum and the newly characterized homologs from its closely related orchid species Phalaenopsis equestris and Apostasia shenzhenica, along with the well-investigated homologs from Arabidopsis thaliana and Oryza sativa. Public RNA-seq data were investigated to analyze the expression patterns of D. catenatum core JA pathway genes in various tissues and organs. Transcriptome analysis of MeJA and S. delphinii treatment showed exogenous MeJA changed most of the expression of the above genes, and several key members, including DcJAZ1/2/5 and DcMYC2b, are involved in enhancing defense ability to S. delphinii in D. catenatum. Conclusions The findings indicate exogenous MeJA treatment affects the expression level of DcJAZ1/2/5 and DcMYC2b, thereby enhancing D. catenatum resistance to S. delphinii. This research would be helpful for future functional identification of core JA pathway genes involved in breeding for disease resistance in D. catenatum.

First Report of Athelia rolfsii Causing Southern Blight on Sarcandra glabra in China

Sarcandra glabra, belonging to the family Chloranthaceae, is a Chinese medicinal plant. The whole dry plant can be used as a medicine; it is rich in bioactive phytochemicals that possess anti-bacterial, anti-inflammatory, anti-oxidant, and anti-tumor properties (Xie et al. 2020). The current market price of S. glabra is around US$5/kg, and the annual demand is 3 500 000~4 000 000 kg in China (Pan et al. 2007). To meet consumer demand for safe and high-quality herbal products, the artificial cultivation of S. glabra has been vigorously promoted. In 2020, it was observed that a plant disease affected S. glabra growth in Hunan province. The disease symptoms included constriction at the base of the stem, with decay and a white mycelium covering. The plants finally died with a disease incidence ranging from 15% to 20%. Using our previously published methods (Yi et al. 2019), one fungal isolate was isolated from the cultured symptomatic stem tissue on potato dextrose agar (PDA) medium and was named as Kb. The isolate was subsequently transferred into 70% glycerol for preservation. The Kb colony varied in color from white to light yellow. The septate hyphae grew rapidly on PDA medium, at approximately 25 mm/day, at 28 °C. On the fifth day, rhizomorphs were formed at the edge and on the center of the PDA plate. On the sixth day, sclerotia developed into a rapeseed shape (d = 1.2~2.3 mm) with a smooth surface, and with white, yellow, or chestnut brown coloring. Morphologically, Kb was similar to Sclerotium rolfsii (Sun et al. 2020). Vigorously growing aerial hyphae were selected for molecular identification. The internal transcribed spacers (ITS) were amplified using the primer pairs ITS1/ITS4 (Glass et al. 1995). BLAST searches against Genbank indicated that Kb’s ITS sequence shared 97% similarity with that of Athelia rolfsii (MN696630.1). Based on morphological and molecular characteristics, Kb was identified as A. rolfsii. The sequence was deposited in GenBank (MW288292). Pathogenicity tests were carried out using the following procedures. Three healthy S. glabra seedlings were inoculated at the stem base with a PDA plug (5 mm in diameter) covered with 5-day-old fungal mycelium cultured at 28 °C, while the remaining three seedlings were inoculated with distilled water only, as the control. Plants were incubated in a greenhouse at 28 °C. At 7 days post inoculation, the inoculated sites infected with the putative pathogen displayed identical constrictions as previously observed in the field. In contrast, the controls remained symptomless. The pathogen was reisolated from these infected seedlings, and its culture showed the same morphological and molecular traits as the original isolates. No pathogens were isolated from the control plants. Pathogenicity tests were repeated three times. Koch’s postulates were fulfilled. Although S. rolfsii has been previously reported to cause Southern Blight on mung bean crops in China (Sun et al. 2020), this is the first report on A. rolfsii causing similar symptoms of Southern Blight on S. glabra in Hunan Province, China. Identification of the pathogens causing each disease is important for the development of effective disease management strategies and for extensive artificial cultivation.

Baseline sensitivity and control efficacy of strobilurin fungicide pyraclostrobin against Sclerotium rolfsii

Sclerotium rolfsii is a fungi pathogen of southern blight with broad host range. The QoI fungicide pyraclostrobin was officially approved for controlling many diseases in 2015. In this study, baseline sensitivity of S.rolfsii to pyraclostrobin was established by measuring the 50% effective concentration (EC50) values of 155 isolates of S.rolfsii. The EC50 ranged from 0.0291 to 1.0871 ug/ml with the mean EC50 values of 0.4469 ± 0.2490 ug/ml (mean ± SD). As preventive fungicide in vitro and field experiment, pyraclostrobin preventive efficacy reached 90% and 80%, respectively, which were much higher than that of control agent carbendazim. Curative efficacy of pyraclostrobin was significantly lower than its preventive efficacy. Pyraclostrobin at 0.1, 0.5, and 2 μg/mL significantly reduced the number of sclerotia produced on potato dextrose agar (PDA) medium, but had no significant influence on their total weight. Pyraclostrobin had no significant influence on mycelial cell membrane permeability, but it significantly reduced oxalate secretion and protein synthesis of S. rolfsii. Our findings are of great significance for resistance monitoring of S. rolfsii and also provide new insight into action mechanism of pyraclostrobin against S. rolfsii.

Role of Chitosan in Disease Suppression, Growth and Yield of Carrot

An attempt was made for controlling of Rhizoctonia canker caused by Rhizoctonia solani and southern blight caused by Sclerotium rolfsii in pot and field experiments under inoculated condition and also to increase the growth promoting factors and yield of carrot through the application of chitosan. Before setting the experiments in the field, laboratory experiments were carried out to select virulent isolates of R. solani and S. rolfsii and effective dose of chitosan on mycelial growth inhibition of virulent isolates of test pathogens. In the pathogenicity test, R. solani isolate R-1 and S. rolfsii isolate S-1 were found to be the most virulent against carrot seedlings. In vitro application of 1.0% chitosan was found to inhibit 100% mycelial growth of both tested pathogens. The field experiment was laid out following randomized complete block design with four treatments, where no treatment was done in T1, pathogen was inoculated in T2 and seed treatment and soil amendment with 1.0% chitosan was done in T3 and T4, respectively, in pathogen inoculated condition. Application of 1.0% chitosan as seed treatment or soil amendment significantly reduced post-emergence seedling mortality, incidence of diseases and enhanced seedling growth and also yields of carrot. On the contrary, post-emergence seedling mortality, incidence of Rhizoctonia canker and southern blight of carrot were highest in treatment T2 where soil was inoculated with pathogens. Chitosan could be used as an alternative of fungicide to suppress Rhizoctonia canker and southern blight in sustainable agriculture and improvement the yield of carrot.